INTERNATIONAL JOURNAL OF AGRICULTURE & BIOLOGY ISSN Print: 1560–8530; ISSN Online: 1814–9596 14–453/2015/17–3–515–522 DOI: 10.17957/IJAB/17.3.14.453 http://www.fspublishers.org

Full Length Article

Production and Characterization of from Streptomyces luteogriseus R10 Isolated from Decaying Wood Samples

Magda Mohamed Aly1, 2, Sanaa Tork1, 3, Saleh Mohamed Al-Garni1* and Saleh Abdulla Kabli1 1Biology Department, Faculty of Science, King Abdulaziz University, Saudi Arabia 2Botany Department, Faculty of Science, Kafr El-Sheikh University, Egypt 3Microbial Genetics Department, National Research Center, Dokki, Cairo, Egypt *For correspondence: [email protected]

Abstract

Microorganisms are the richest source of phytase which catalyze hydrolysis of phytate to myo-inositol and phosphate. 50 Actinomycete isolates were isolated from heated soil, sand, wastewater, animal faces and some plant ecological wastes on Starch nitrate agar containing 15 µg / mL of antibiotics (Tetracycline + Amphotericin B, w/w). Out of 50 Actinomycetes isolates, 20 isolates (40%) produced extracellular phytase on solid medium containing wheat bran as carbon source. In liquid medium, the phytase activity was measured as U/mL and the most active isolate in phytase production was R10. Using morphological, physiological and biochemical studies, it was identified as Streptomyces sp. Using 16S rDNA analysis, it was identified as S. luteogriseus R10. Growth in medium containing 1% Na-phytate (pH 6.5) at 40°C for 7days increased phytase production. The maximum phytase activity was achieved using wheat bran, straw, rice husk and hay after seven days of incubation at 40°C but low activity was obtained using Sawyer and baggage as a carbon source. The molecular weight of the purified phytase is 65 kDa and it exhibits optimum activity at pH 5 and 45°C. All tested metal ions at 10 mM enhanced phytase activity except Ba2+, Co2+, Cu2+, Ag, Fe3+ and Hg2+. Improvement of phytase production was carried out using protoplast fusion between S. luteogriseus R10 and S. niveus MM1. Fusant F7 was the best phytase producer (3 time higher) compared to its parents. © 2015 Friends Science Publishers

Keywords: Phytase; Streptomyces; Protoplast; Fusant; Phytic acid

Introduction low level of phytase in primitive natural microbes.

For humans and animals, legumes, cereals and oilseed crops Materials and Methods are the main source of nutrients, which contain phytic acid as storage form of phosphorus (Reddy et al., 1989). To Bacterial Isolation satisfy phosphorus requirement, inorganic phosphate is added to monogastric animals diets because they cannot Soil, sand, plant materials, wastewater and plant waste metabolize phytic acid present in their diet. In monogastric products from farms in Huda Al Sham, Saudi Arabia were animals, phytic acid is chelating various metal ions collecting and transported in sterile plastic bags to the including calcium, copper and zinc (Graf, 1983). Therefore, Microbiology lab. Serial dilutions were prepared and about phytic acid hydrolysis into less-phosphorylated myo-inositol 0.1 mL of each sample was spread on starch nitrate medium derivatives using phytase is of great interest. Phytase can be (Shirling and Gottlieb, 1966) which composed of g/L: 20 used to improve the nutritional value of feed and/or to Starch, 1.0 KH2PO4, 0.5 MgSO4.7H2O, 0.01 FeSO4.7H2O, decrease the amount of phosphorus excreted by animals. 3.0 CaCO3, 2.0 KNO3, 0.5 NaCl and 1 mL trace salt Phytase enzyme produced by bacteria is extracellular solution) and for solid medium preparation, 20 g/L agar was which are more appropriate than the intracellular added. Filter sterilized mixture of phytase produced by yeast in breaking down phytic acid Tetracycline+Amphotericin B (w/w) was added at (Konietzny and Greiner, 2004). The aim of the present concentration 15 µg/mL to the prepared medium (Aly et al., study was isolation and identification of thermo-stable 2011b). Incubation of the plates was at 37°C for 7 days and phytase producing bacterium and factors affecting the purified colonies were streaking on ISP2 medium production were studied. Phytase was purified and composed of g/L: 4 yeast extract, 4 glucose, 10 malt characterized. Enhancement of enzyme production was extract, 2 CaCO3 and 20 agar and maintained at 4°C carried out using protoplast fusion to solve the difficulty of until used.

To cite this paper: Aly, M.M., S. Tork, S.M. Al-Garni and S.A. Kabli, 2015. Production and characterization of phytase from Streptomyces luteogriseus R10 isolated from decaying wood samples. Int. J. Agric. Biol., 17: 515‒522

Aly et al. / Int. J. Agric. Biol., Vol. 17, No. 3, 2015

Bacterial Growth and Screening Conditions precipitate at 10,000 rpm was dissolved in phosphate buffers (pH 7), dialyzed against the same buffer for 2 days Several Actinomycete isolates were screened on modified (El-Sabbagh et al., 2003), concentrated under vacuum, LB with wheat bran medium (LBWB medium). LB medium applied to a column (30 × 1.5 cm) of diethyl aminoethyl composed of (g / L) 10 g tryptone, 10 g NaCl and 5 g yeast cellulose (DEAE cellulose) and eluted using 1 M NaCl in extract in addition to 50 g of wheat bran, pH was adjusted to phosphate buffer (80 mL / h). The eluent was collected in 5 pH 7.0. Agar (20 g / L) was added, when solid agar medium mL fractions. The active fractions with phytase activity was needed. In liquid broth medium, the bacterial isolates were collected and concentrated under vacuum. The with phytase activities were cultivated in 50 mL LBWB concentrate was applied to carboxymethyl-cellulose broth medium in 250 mL Erlenmeyer flasks. Each flask was followed by Sephadex G75 column and elution was carried inoculated with 2mL (4×106 CFU∕mL) of bacterial out using phosphate buffer. The active fraction was suspension, previously grown at 37°C for 2 days at 200 rpm collected, lyophilized and was analyzed. Gel electrophoresis in starch nitrate broth medium. The inoculated flasks were was carried with 15% SDS polyacrylamide electrophoresis maintained for 5 days at 37°C on a rotary shaker (200 rpm). at room temperature where 20‒30 μL (40 μg / well) from After centrifugation at 10,000 rpm for 15 min, cell-free the protein standard (Merck) were applied and gel was supernatant was for phytase assay. stained with Coomassie brilliant blue R-250.

Phytase Assay Characters of the Pure Phytase Enzyme

Cell-free supernatant was used for determination of phytase Using the standard phytase assay conditions (Chi et al., activity by measuring the amount of phosphorous released 2008), effect of different pH values, 3‒9, on phytase was from Na-phytate, which develops a color with ammonium determined after the pure enzyme suspension in 0.2 M molybdate. The color density was quantified acetate buffer (pH 3‒6) or 0.2 M Na2B4O7.10 H2O / H3BO3 spectrophotometrically at 700 nm (Chi et al., 1999). Under buffer (pH 7‒10). Effect of temperature ranged from 20- assay conditions, one phytase unit was the amount of the 70ºC on the purified was determined in the enzyme, released of 1 nM of inorganic phosphate / min. selected buffer. The pre-incubated enzyme at 37°C was used as a reference to calculate activity. Effect of some additives Optimization of Phytase Production Process incorporated in the reaction mixture including some metal ions and EDTA on the enzyme assay was studied and the In liquid medium, the effect of different factors on growth relative activities were compared with the activity obtained (optical densities at 540 nm) and phytase production was for control (without additive) (El-Sabbagh et al., 2003). determined (El-Sabbagh et al., 2003). LB broth medium with different concentrations of Na-phytate was prepared. Characterization of the Selected Actinomycete Isolate The inoculated flasks were incubated at 37ºC and 200 rpm (Taxonomical Studies) for 5 days. Effect of different incubation temperatures (20- 50°C) and medium pH (6.0-8.0) were determined after 5 The selected phytase producing actinomycete isolate was day of growth in LB medium with 1% Na-phytate. At the characterized and identified. It was grown on starch nitrate end of growth period, growth and phytase were measured. agar medium for fresh prepared culture. Gram and Effect of different incubation periods ranging from 1 to 7 endospore stains were carried out and bacterial morphology days of growth in modified LB medium (pH 6.5) at 40°C was conducted using light and electron microscopy (XL30- and 200 rpm was determined. All the experiments were ESEM environment scanning electron microscopy). carried out in triplicate and averages were reproduced. Physiological and biochemical characters in addition to chemical analysis of the whole cell sugar composition and Phytase Production Using Various Waste Products type of the diaminopimelic acid isomer were determined as described by Aly et al. (2011a, 2012) and Hasegawa et al. Some agriculture waste products were collected, dried, (1983), respectively. Cell fatty acids and phospholipids were powdered and sterilized using the autoclave. They were extracted and determined using gas chromatography and used as carbon source as described by Lanciotti et al. (2005) two-dimensional thin-layer chromatography (Butte, 1983; and phytate hydrolysis was determined after 7 days of Hoischen et al., 1997). incubation at 40°C and 200 rpm. Phylogenetic Analysis of 16S rDNA Sequence Enzyme Purification and Molecular Weight Determination QIAamp DNA Mini Kit was used for genomic DNA extraction of the selected isolate R10. The forward primer 5' Proteins in the culture filtrate were collected after 80% AGTTTGATCATGGTCAG-3' and reverse primer 5' Ammonium sulfate precipitation at 4°C. The collected GGTTACCTTGTTACGACT 3' were designed (Weisberg

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Improving Phytase Production

Improving phytase production using protoplast fusion (Yari et al., 2002; Aly et al., 2011a, b) between Streptomyces R10 Fig. 1: Screening of some Actinomycete isolates on and S. niveus (MM1) which was highly resistant to NaCl LBWB medium for phytase production after 5 days of (Aly et al., 2003) was carried out. On Mueller Hinton growth medium, antibiotic pattern using paper disc diffusion assay for the two tested bacteria were as the following: Streptomyces R10 was strep.- tet. + and S. niveus was strep.+ tet-. The two species were grown at 37°C for 24 h in starch nitrate broth medium (Shirling and Gottlieb, 1966). From the previous culture, 1 mL was mixed with 10 mL of a starch nitrate broth with 0.5% (w / v) glycine in100 mL conical flask and the flasks were incubated at 30°C overnight. The growth was collected by centrifugation (5,000 rpm for 15 min) and sonicated for 3min for Fig. 2: The selected Actinomycete isolate R10, A: under protoplast formation (Matsushima and Baltz, 1985). The scanning electron microscope (x 20 000), B: under light percentages of real protoplasts for each species were microscope x1000, C: after growing on Oat meal agar medium calculated (Aly et al., 2011b) and the obtained protoplasts for 10 days at 37°C were mixed gently in 3 mL of Modified R2 sterile medium containing the appropriate concentration of tetracycline microscope using oil emersion lens. It was belonging to (1µg / mL) and streptomycin (5 µg / mL). After 7 days of filamentous Gram + ve bacteria with gray color (Fig. 2). incubation at 30°C, the obtained fusants were picked and The isolate R10 was identified according to morphological, maintained on the same medium. physiological and biochemical characters (Table 2, 3, 4 and 5). Cell wall composition and the characteristics sugars, Statistical Analysis lipids and fatty acids were determined (Table 6). Presence of L-isomer of diaminopimelic acid (L-DAP) and glucose Mean of three replicates and standard deviations were indicated a wall chemotype IV and whole cell sugar pattern recorded. Data were statistically analyzed and difference as type A, while analysis of phospholipids indicated between mean values was determined using Student’s t-test. phospholipids type PII. Saturated fatty acids with no The differences were significant when P<0.05. mycolic acids were detected using gas chromatograpy. The phylogenetic tree based on 16S rDNA sequence using Results neighbor joining tree method was drowning (Fig. 3). Phytase production or / and bacterial growth varied This research aimed to isolate many Actinomycete isolates, with phytate concentration, initial pH of the medium, producing phytase, which can hydrolyze some agricultural incubation temperature and incubation period. The best wastes. In this connection, 50 bacterial isolates with phytate recovery was 1.0% (Fig. 4). The effect of different colony colors and shapes were obtained from soils, temperature and pH on phytase production was showed in sand and plant materials in addition to ecological wastes on Fig. 5 and 6, where incubation at 40°C and initial pH 6.5 starch nitrate agar-containing antibiotic. All the isolates recorded maximum phytase production. However, phytase were grown on LBWB agar medium and 20 isolates out of production dropped significantly at pH 9.0 and no 50 grow well on the previous medium hydrolyzing the production was observed at pH 9.5. The maximum phytase phytic material by phytase, which was detected as pale clear production was recorded after 7 days of growth at 40°C zone around the colony (Fig. 1). Diameter of the clear zone (Fig. 7). Different waste product including hay, straw, and was differed for each organism. Six phytase producing bran (Fig. 8) were used as substrate (1% w / v) for phytase bacterial isolates were selected and grown in liquid broth production. Among all the substrates, the maximum phytase medium (Table 1). activity was observed with bran and straw. The lowest In liquid medium, the most active isolate in phytase production was observed for sawyer and baggage. production was isolate R10, which was isolated from The selected isolate R10 was grown using the best decaying wood sample on SN agar with antibiotic. The conditions of phytase production. The enzyme was morphology of the phytase-producing bacterium and Gram precipitated and purified using different column reaction were determined after examination with light chromatography and profile of elution was determined.

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Table 1: Source, colony color and phytase production by the most active Actinomycetes in phytase production

Bacterial isolate Source of isolation Colony color Phytase production On solid medium In liquid medium Presence of clear zone activity (U /mL) R1 Wastewater White + 1.0±0.09 R10 Decaying wood Gray +++ 1.9±0.05 R24 Contaminated soil White ++ 1.7±0.03 R36 Contaminated soil Yellow + 1.7±0.01 R47 Contaminated soil Pink + 1.1±0.04 R 49 Animal feces Yellow + 1.1±0.05 +: Moderate production, ++: High production, +++: Very high production

Table 2: Cultural characteristics of the Actinomycete isolate R10 grown on different agar medium at 30°C

Agar medium Growth Color of aerial mycelium Color of substrate mycelium Presence of soluble pigment Glycerol-asparagine agar (ISP-5) Moderate Dark gray Yellow + Glucose Asparagine agar Heavy Dark gray Yellowish gray + In-organic salts-starch iron (ISP-4) Moderate Pale gray Yellowish-white + Tyrosine agar (ISP-7) Scanty Yellowish gray Pale yellow + Yeast extract-malt extract (ISP-2) Moderate Yellowish brown Yellow + Oatmeal agar (ISP-3) Moderate White Pale yellow + +: Soluble pigment present

antibiotics resistance profiles (10 μg / mL tetracycline and 400 μg / mL streptomycin), was carried out using PEG 6000.The regenerated protoplast percentage of the two species was 88% and 80%, respectively. Ten recombinant fusants were obtained and fusant F7 showed higher phytase production compared to the two wild types (about 3 fold increases) (Table 7).

Discussion

Different actinobacteria were obtained from the collected samples on agar medium containing antibiotics. Addition of antibiotics to growth medium or samples heating prevent the growth of unwanted bacteria and allow antibiotic resistant actinobacteria to dominate (Velho-Pereira and Kamat, 2011). Similarly, on modified glycerol arginine agar, an initial screening was performed to isolate common Actinomycetes and on modified medium to isolate rare Actinomycetes (Ghorbani-Nasrabadi et al., 2012). Fig. 3: Phylogenetic tree based on 16S rDNA sequence In solid LBWB agar medium containing wheat bran as comparisons of Streptomyces R10, using neighbor inducer, all Actinomycete isolates were screened for phytase production. As it is well known, the phytase enzyme was joining tree method, maximum sequence difference inducible, and the presence of phytate, wheat bran or some =0.002 other inducer in the medium is necessary for enzyme The active fractions that showed the maximum phytase formation (Tambe et al., 1994; Konietzny and Greiner, activity were collected, lyophilized and used for enzyme 2004). In bacteria, induction and expression of phytase are characterization and molecular weight determination. regulated and are not controlled uniformly among different Phytase molecular weight was 65 kDa, detected using gel bacteria. Phytase may not require during balanced bacterial electrophoresis (Fig. 9) and exhibits optimum activity at pH growth but synthesized under energy and / or nutrient 5.0 and 45°C (Fig. 10). Phytase activity was significantly limitation (Konietzny and Greiner, 2004). In this work, 40% affected by most of the ions tested and all these ions at 10 of the screened actinobacteria were phytase-producing and mM acted as enhancer for the phytase activity except Ba2+, this activity was detected as clear zones accompanying the Co2+, Cu2+, Fe2+, Fe3+, Li+ and Hg2+ (Table 6). growth in solid agar. Similarly, 46.3% of the Actinomycete Protoplast fusion between the identified S. isolates had phytate-degrading capacity (Ghorbani- luteogriseus R10 and S. niveus MM with two different Nasrabadi et al., 2012). Out of 21 bacterial isolates from

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Table 3: Morphological character of the selected isolate R10

Tested character Results Gram stain Gram positive Source of isolation Decaying wood Motility of spore Absent Shape of spore Cylindrical (5-6 and, 6-9 µm) Spore chain Spiral chain Spore Surface Hairy Number of spore / chain 5-20 Fig. 4: Effect of different concentration of phytate on Aerial and substrate hyphae Well developed growth and phytase production by the selected strain of Zoospore, sporangium, sclerichia, Absent fragmented mycelia Actinomycetes isolate R10

Table 4: Physiological characteristics of the isolate R10

Character Reaction Character Reaction Melanin pigment production +ve Tolerance to NaCl 5-10% Enzyme activities: pH range 6-10 Proteolysis +ve Growth temperature: 15-45ºC Lecithinase -ve Resistance to antibiotic Lipolysis +ve Penicillin Chitinase +ve Cephalosporine +

Gelatinase +ve Kanamycin + Pectinase +ve Rifampin + Fig. 5: Effect of different temperature on growth and H2S Production -ve Tetracycline + phytase production by the selected strain of -ve: negative results, +ve: positive results, ++: Growth, +: Resistance Actinomycetes R10 water habitats, eight showed phytase production by clear 2012), was used. The 16S rDNA sequence reported that zone formation around growth on solid agar medium isolate R10 was closely related to S. luteogriseus by 93% (Shamna et al., 2012). Furthermore, 67 isolates of soil and can be identified as S. luteogriseus R10. actinomycetes were potentially producing extracellular The knowledge on the participation and role of phytate-degrading activity and two isolates of the genus Actinomycetes in hydrolysis of phytate and organic Streptomyces were the most active in phytase production phosphorylated compounds is extremely limited and is (Ghorbani-Nasrabadi et al., 2012). strongly dependent on the Actinomycete strain as well as Phytase detection was obtained using plate-clearing media composition. Furthermore, the importance of technique and / or measuring the enzyme activity in liquid Actinomycetes in dephosphorylation of soil organic medium. For bacteria, growth on agar medium is usually compounds needs to be elucidated in further studies. The easier than in a liquid broth medium (Choi et al., 2001), results of Ghorbani-Nasrabadi et al. (2012) showed thus, no further assessment was conducted for isolates that Actinomycetes as a source of phytase. On contrast, it was could not grow or showed weakly growth on solid agar clear that production of were characterized from medium containing wheat bran as source of phytate. The some Gram-negative and positive bacteria including lack or weak phytase activity in some Actinomycete isolates Klebsiella, Enterobacter, Bacillus and Pseudomonas (Yoon may be due to a loss of trait in the isolate or the used culture et al., 1996; Greiner et al., 1997; Richardson and Hadobas, conditions were not effective in inducing phytase. Hence, at 1997; Kerovuo et al., 1998; Richardson et al., 2001). this point, it is unclear whether phytate hydrolysis is a Although, phytases have found in some animal tissues and common trait within the Actinomycete group. Generally, in plants, phytases from bacteria have many commercial some bacterial enzymes are mostly cell associated, whereas applications due to substrate specificity, catalytic efficiency the phytases obtained by fungi are extracellular. Concerning and resistance to proteolysis (Konietzny and Greiner, 2004). the isolate R10, phytase production was extracellular in the In liquid medium, maximum enzyme production by S. culture filtrate and it was identified using morphological, luteogriseus R10 was occurred using LB medium physiological, biochemical analysis in addition to 16S continuing 1% Na-phytate as inducer, incubation of flasks at rDNA analysis. According to morphological description 40°C, initial pH 6.5 and 200 rpm after 7 days. (Pridham and Tresner, 1974), biochemical comparison and Cladosporium sp. FP-1 showed maximum phytase physiological analysis of genus Streptomyces with other production in a medium containing 1.0 g phytate (Quan et described isolates (Williams et al., 1989), the isolate R10 al., 2004). Temperature is one of the most critical belongs to the genus Streptomyces. Identification was made parameters to be controlled in any bioprocess and the using 16S rDNA, a powerful tool for deducing evolutionary optimum temperature for production of phytases by many relationships and phylogenetic among eukaryotic microorganisms was 25-37°C (Vohra and Satyanarayana, organisms, bacteria and archaebacteria (Olmezoglu et al.,

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Table 5: Utilization of different carbon and nitrogen sources using the selected isolate R10

Carbon source Utilization Nitrogen source Utilization Positive control (glucose) ++ NaNO3 ++ Negative control -ve NH4NO3 ++ D-mannitol ++ KNO3 ++ Glycerol ++ NH4OH ++ Raffinose -ve NH4Cl ++ Fig 7: Effect of different incubation periods on growth D – galactose -ve NaNO2 -ve Sucrose ++ Phenyl alanine + and phytase production by the selected strain of Fractose ++ Valine + Actinomycetes R10 D-xylose ++ Peptone +

Fig. 8: Growth and phytase production of the selected Fig. 6: Effect of different initial pH value on growth and strain of Actinomycetes isolate R10using different phytase production by the selected strain of waste products (10 g/L) as carbon source Actinomycetes R10 Some bacterial phytases with pH optimum of 6.0 to 2003). At pH 6.5, maximum phytase production was 8.0 benefits in poultry feed additives where their pH recorded and increasing medium pH decreased the activity optimum was close to the physiological pH of the poultry of the enzyme due to charges on the amino acids within the crop (Kim et al., 1998; Choi et al., 2001). In contrast, and no formation of the enzyme-substrate purified phytase from Bacillus sp. DS11 had molecular complex. Similar results were obtained for phytase of weight of 44 kDa by SDS-polyacrylamide gel Pseudomonas spp, (Sasirekha et al., 2012). For electrophoresis and optimum temperature of 70°C in Enterobacter sp., the optimum phytase production was at presence of calcium ions but its activity was greatly pH 5.5 after 3 days of growth at 37°C (Yoon et al., 1996). inhibited by metal ions such as Cd2+, Mn2+ and EDTA (Kim Phytase of Streptomyces was very specific for phytate and et al., 1998). Moreover, phytase with lower MW of 32.6 efficiently hydrolyzed phytate of wheat bran, hay, straw, kDa, optimum temperature of 40°C, optimum pH of 3.5, rice hush sawyer and baggage. Similar results were obtained stimulated by dithiothreitol and 2-mercaptoethanol, and by Kim et al. (1998) where, phytase was very specific for inhibited by Ba2+ and Pb2+ was obtained (Quan et al., 2004). phytate and had little activity on other phosphate esters and Phytases from Enterobacter sp. and Bacillus subtilis were efficiently hydrolyzed phytate in oat flour rice and wheat. inhibited by 1 mM EDTA (Yoon et al., 1996; Kerovuo et Using the best conditions for phytase production, the al., 1998). On contrast, Greiner (2004) found that phytase enzyme was collected and purified using different column was not inhibited by EDTA at concentration of 1 mM chromatography. After purification, phytase has molecular whereas phytase from A. niger van Teighem was enhanced weight of 65 kDa and its activity was not significantly affected by most of the ions tested. However, EDTA, Ag+, by EDTA (0.1-2.0 mM) for about 50% (Vats and Banerjee, Cd2+, Hg2+, Cu2+ inhibit the phytase activity. Similarly, two 2002). The maximal phytase activity of Enterobacter sp. 4 species belonging to the genus Streptomyces produced was observed at pH 7.0-7.5 and at 50°C but above 60°C, the enzyme activity was gradually lost and was inhibited by phytase with optimum temperature of 55°C and 37°C and 2+ 2+ 2+ 3+ optimum pH values of 5 and 7 (Ghorbani-Nasrabadi et al., each addition of 1 mM Zn , Ba , Cu , Al and EDTA 2012). The purified enzyme of Bacillus had maximal (Yoon et al., 1996). phytase activity at pH 7 and 55°C and required calcium for Furthermore, enhancement of phytase production its maximum activity but was readily inhibited by EDTA using protoplast fusion was carried out between the (Kerovuo et al., 1998). The optimum pH of phytases from identified Streptomyces and Streptomyces niveus, which E. coli, Klebsiella or Aspergillus were in the range 4.5-5.5 showed different antibiotic resistance profiles. Percentage of (Greiner et al., 1993; Wyss et al., 1999, Sajidan et al., 2004; successfully regenerated protoplasts of the two used Elkhalil et al., 2011). In contrast to those phytases, Bacillus Streptomyces was 88% and 80%, respectively. Out of 10 phytase displayed a narrow pH optimum between 7.0 and recombinant fusants obtained, one (Fusant F7) showed 7.5 (Kerovuo et al., 1998; Kim et al., 1998). higher phytase production compared to both parents (3

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Table 6: The biochemical tests (sugar, amino acid, phospholipids, and fatty acid composition of the cell wall or cell hydrolysate) of the isolate R10

Type of the reaction Results Sugar in the cell hydrolysate (glucose) + Amino acids in the cell wall (diaminopimelic acid, DAP) L-form Phospholipids (phosphatidylethanolamine and phosphatidylinositolmannoside) + Fatty Acids Iso. and Antiso fatty acid

Table 7: Effects of different metal ions and chemicals on phytase activity

Metal Relative activity (%) CaCl2 120 MgCl2 120 MnCl2 120 ZnSO4 100 CuSO4 30 FeSO4 90 LiCl 55 AgNO3 40 HgCl2 20 CoCl2 57 BaSO4 90 EDTA 50

Table 8: Phytase production using two tested Streptomyces isolates and their fusants Fig. 9: SDS-PAGE profile of purified phytase, Lane A: Used bacteria Phytase activity Used bacteria Phytase activity standard protein marker, lane B: purified phytase 2 (U/mL) × 102 (U/mL) × 10 Streptomyces niveus 0.40 Fusant F5 0.0 and many industrial applications. The purified phytase Streptomyces R10 (control) 1.7 Fusant F6 0.08 enzyme has 65 kDa and was stable at 45°C and pH5. Fusant F1 0.09 Fusant F7 7.90* Genetic improvement using protoplast fusion enhanced Fusant F2 0.80 Fusant F8 0.80 Fusant F3 0.04 Fusant F9 0.70 phytase production. Fusant F4 0.05 Fusant F10 5.0* *: significant difference at p< 0.05 Acknowledgements fold increase). The previous results were in agreement with This project was funded by the Deanship of Scientific those obtained by many authors (Chassy, 1987; Kanatani Research (DSR), King Abdulaziz University, Jeddah, Saudi et al., 1990; Ward et al., 1993) who used protoplast Arabia under grant no (1432/247/287). The authors fusion in genetic manipulation and bacterial improvement. therefore acknowledge with thanks DSR technical and After protoplast fusion between Streptomyces cyaneus and financial support. Streptomyces griseoruber, Teeradakorn et al. (1998) isolated new fusants with rearrangement in their genetic References materials and produced high levels of xylanase. Lin et al. (2007) isolated several fusants with increased lipase activity Aly, M.M., S. El-Sabbagh, W. El-Shouny and M.K. Ebrahim, 2003. (317%). Through protoplast fusion, fibrinolytic enzyme Physiological response of Zea mays to NaCl stress with respect to production from Bacillus was improved 4-5 time compared Azotobacter chroococcum and Streptomyces niveus. Pak. J. Biol. Sci., 6: 2073–2080 to wild type (Liang and Guo, 2007). Moreover, Aly et al. Aly, M.M., S. Tork, S.M. Al-Garni and L. Nawar, 2012. Production of (2011a, 2012) used the previous technique to enhance lipase from genetically improved Streptomyces exfoliates LP10 chitinase and lipase production by Streptomyces and mutate isolated from oil contaminated soil. Afr. J. Microbiol. Res., 6: 1125– 1137 Bacillus to enhance chitinase production. Aly, M.M., S. Tork, S.M. Al-Garni and S.A. Kabli, 2011a. Chitinolytic enzyme production and genetic improvement of a new isolate Conclusion belonging to Streptomyces anulatus. Ann. Microbiol., 61: 453–461 Aly, M.M., S.A. Kabli and S.M. Al-Garni, 2011b. Production of Decaying wood contained many actinomycete isolates with biodegradable plastic by filamintous bacteria isolated from Saudi excellent phytase production and S. luteogriseus R10 was Arabia. J. Food Agric. Environ., 9: 751–756 Butte, W., 1983. Rapid method for the determination of fatty acid profiles the most active isolate which can degrade some plant waste from fats and oils using trimethyl sulphonium hydroxide for products including hay, straw and bran using phytase. transesterification. J. Chromatgr., 261: 142–145 Optimization of bacterial growth conditions enhanced Chassy, M.B., 1987. Prospects for the genetic manipulation of Lactobacilli. phytase production which can be used in bioremediation FEMS Microbiol. Lett., 46: 297–312

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